Screening method for ligands with class II PI3 kinase modulating activity

ABSTRACT

A novel human class II PI3 kinase including the sequence of the isolated nucleic acid molecule which encodes the kinase and the encoded amino acid sequence. The new human PI3 kinase is termed PI3K-C2α and has unique biochemical properties that characterize it and distinguish it from previously known PI3 kinases. These include, among other things, resistance to the PI3 kinase inhibitors Wortmannin and LY294000, the lack of a p85 binding site, a divergent amino terminus, and the absence of a polyproline motif which is typical of previously known type II PI3 kinases.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a division of co-pending U.S. patent application Ser. No. 09/355,160, now U.S. Pat. No. ______, which is the U.S. national stage of international patent application no. PCT/GB98/00244, filed Jan. 27, 1998, designating the United States of America. Priority is claimed based on United Kingdom patent application Ser. No. GB 9701652.1, filed Jan. 28, 1997.

BACKGROUND OF THE INVENTION

[0002] The invention relates to a novel class II PI3 kinase, termed PI3K-C2α, and in particular the isolation thereof having regard to its relevant sequence structure and/or biochemical characteristics; means used in the isolation or production thereof; antibodies adapted to bind thereto; and assay kits relating thereto.

[0003] Phosphoinositides have been implicated in a variety of cellular processes as diverse as vacuolar protein sorting (1,2), cytoskeletal remodelling (3) and mediating intracellular signalling events through which growth factors, hormones and neurotransmitters exert their physiological effects on cellular activity, proliferation and differentiation (4,5,6).

[0004] Recently a family of proteins have been cloned and characterised and shown to be enzymes catalysing the addition of phosphate to inositol. Eukaryotic cells contain a variety of inositol derivatives phosphorylated to different extents. PtdIns(3)P is constitutively present in eukaryotic cells and its levels are constant upon extracellular stimulation. PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ (7,8) are virtually absent in resting cells but are rapidly induced upon stimulation with a variety of ligands. The enzymes catalysing these reactions are phosphoinositide lipid kinases (hereinafter called PI3 kinases). A brief overview of the current data in relation to PI3 kinases classifies these enzymes into three distinct groups being designated to an individual class by their in vitro substrate specificity, biochemical characteristics and, in examples where a definitive function has been assigned, the nature of the biochemical activity regulated by the specific kinase.

[0005] PI3 kinase class 1 polypeptides have a broad spectrum activity, phosphorylating inositol lipids PtdIns, PtdIns(4)P and PtdIns(4, 5)P₂. Class I kinases are subdivided into Class IA and IB. Class IA polypeptides include p110α (9), p110β (10) and p110δ (11) which interact physically with the adaptor sub-unit protein p85. Moreover, p110α p100 have a broad distribution in terms of expression pattern. p110δ expression seems to be restricted to white blood cells. Class 1B includes p110γ (12) which functions independently of p85. Although each of these Class 1 kinases catalyse phosphate addition to inositol lipid, the mechanism via which these enzymes are activated and regulated is achieved by different molecular mechanisms.

[0006] Class II PI3 kinases have a restricted substrate specificity phosphorylating PtdIns and PtdIns(4)P but not PtdIns(4,5) P₂. Each of the kinases of this class are characterised by a conserved C2 domain in the carboxyl terminal region of the protein. The presence of conserved motifs within the C2 domain indicates that this region may confer regulation via calcium and/or phospholipid. A comparison of the murine and Drosophila class II kinases mp170 and PI3K_(—)68D respectively reveals a high degree of homology in the kinase domain of these proteins. Significant divergence occurs at the amino terminal regions of these polypeptides suggesting that adaptor proteins interacting with these variable domains may regulate kinase activity. Class II PI3 kinases do not interact with p85.

[0007] The third class of PI3 kinase, class III PI3 Kinase, is related to the S.cerevisiae gene Vps34 (1). This kinase was originally isolated as a gene involved in regulating vesicle mediated membrane-trafficking in yeast. The human homologue of Vps34 is complexed with a ser/thr kinase called Vps15p (14,15). Of the three classes of PI3 kinase this has the most restricted substrate specificity being strictly limited to PtdIns.

SUMMARY OF THE INVENTION

[0008] A novel human class II PI3 kinase is herein described and termed human PI3K-C2α. It is characterised as a class II kinase due to the presence of a conserved C2 domain found in murine and Drosophila class II PI3 kinases (FIG. 2), its apparent lack of a p85 binding site and a substrate specificity limited to PtdIns and PtdIns (4) P (FIG. 4). The polypeptide is unique in that this is the first human class II kinase to be described. It has significant divergence in the amino terminal region of the protein when compared to the mouse homologue of human PI3K-C2α (16). It is also, surprisingly, the first PI3 kinase to be isolated that has resistance to PI3 kinase inhibitors Wortmannin and LY294002 (FIG. 5).

[0009] The use of selected inhibitors has proved extremely useful in analysing intracellular signal transduction cascades. Inhibitors used at low concentrations probably result in the modification of a single protein's function thereby allowing the dissection of single transduction pathways. A good example of this is the use of Pertussis toxin which is a cell permeant agent (17). The agent undergoes endocytosis into intact cells and results in the ADP-ribosylation of specific GTP-binding or G-proteins. This modification uncouples these G-proteins from their receptors therefore interfering with the cell's response to receptor stimuli. Wortmannin is another cell permeant inhibitor (18,19). It is a fungal metabolite and has been shown to have in vivo anti-inflammatory or immunosuppressive effects in animal models. Wortmannin was first shown to inhibit cellular responses to receptor stimulation in neutrophils. The drug inhibited the respiratory burst induced by ligands such as N-formyl-Met-Leu-Phe,(fMLP),C5a,leukotriene B4 or platelet-activating factors. Importantly, Wortmannin failed to inhibit cellular response to TPA suggesting differential responses to the drug. In particular, the fact that the stimulation of calcium mobilising receptors is resistant to Wortmannin suggests that intracellular signalling initiated by these receptors is controlled by a quite separate kinase cascade.

[0010] The identification of PI3 kinases as the target for Wortmannin came from in vitro metabolic labelling of guinea pig neutrophils with ³²p to monitor the uptake of phosphate into phospholipids in the presence of specific kinase inhibitors (20). In control experiments stimulation of fMLP receptors resulted in ³²p labelling of phosphatidic acid and PtdIns(3,4,5)P₃. The presence of Pertussis toxin had no effect on the phosphorylation of these phospholipids. However, pre-incubation of leucocytes with Wortmannin resulted in inhibition of ³²P incorporation into only PtdIns(3,4,5)P₃. Since this is the product of PI3 kinase catalysed reactions it seems likely that Wortmannin was specifically targeting PI3 kinase. Subsequently, Wortmannin has been shown to block a number of physiological processes including many insulin stimulated actions that would result in enhanced glucose utilisation (21). Wortmannin has proved to be an effective inhibitor of mammalian PI3 kinases. To date, no human PI3 kinase has been cloned and shown to be resistant to this agent.

[0011] The isolation and sequencing of an as yet unidentified human PI3 kinase that has significant homology to previously identified murine and Drosophila class II kinases is described (16,22,23). Comparison of the optimal alignment of these aforementioned proteins shows the human protein to be 32.5% homologous with sequences of the Drosophila PI3K_(—)68D and cpk proteins and 90.8% and 90.2% with the murine proteins mp107 and mcpk, FIG. 2. The carboxyl terminal region of the aforementioned proteins have increased homology due to the presence of a conserved C2 domain. This domain is characterised by the presence of motifs likely to be involved in the modulation of kinase activity by calcium and/or phospholipid.

[0012] The amino terminal region of human PI3K-C2α is extended by 176 amino acid residues which are lacking in the murine sequence of mp170. The murine mcpk protein has a 28 amino acid residue deletion in this region that is absent from human PI3K-C2α and a mp170. This divergence in sequence may be explained by the presence of the unique binding sites for adaptor proteins that regulate kinase activity. This is supported by the lack of an apparent p85 binding motif.

[0013] Furthermore, immunofluoresence experiments using a monoclonal antibody to the amino terminal glu-tag of PI3K-C2α reveals a punctate cytoplasmic distribution in Cos cells expressing the recombinant glu-tagged PI3K-C2α protein, FIG. 9. Moreover subcellular fractionation of Monkey Kidney Cos cells shows that epitope tagged PI3K-C2α is tightly associated with phospholipid containing membranes. The nature of this interaction is currently unknown but may be mediated via phospholipid interaction with the C2 domain or alternatively through binding to an additional polypeptide localized to microsomes and plasma membranes.

[0014] The recombinantly produced PI3K-2Cα kinase shows increased resistance to the general PI3 kinanse inhibitors Wortmannin and LY294002, (FIG. 5) To date, aall clones human PI3 kinases have been found to be sensitive to these inhibitors. Studies of the Wortmannin sensitive class I PI3 kinanse p110α identifies residue lys-802 as being the sit of Wortmannin covalent inactivation of kinase activity, a residue near or at the active site and involved in phosphate transfer. This residue is present in all PI3 kinase family members and also in PI3K-2Cα, therefore the resistance to Wortmannin by PI3K-2Cα is not apparent.

[0015] Additionally it will be apparent that alternate methods for use in modulating the activity of PI3K-2Cα exist.

[0016] For example, having regard to the sequence data provided herein it is possible to provide antisense material which prevents the expression of PI3K-2Cα.

[0017] As mentioned above, the invention embraces antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding a PI3K-2Cα protein, to decrease transcription and /or translation of PI3K-2Cα genes. This is desirable in virtually any medical condition wherein a reduction in PI3K-2Cα gene product expression is desirable, including to reduce any aspect of a tumor cell phenotype attributable to PI3K-2Cα gene expression. Antisense molecules, in this manner, can be used to slow down or arrest such aspects of a tumor cell phenotype.

[0018] As used herein, the term “antisense oligonucleotide” or “antisense” describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide with hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and thereby, inhibits the transcription of that gene and/or the translation of that mRNA. The antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence. It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physilogical conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Based upon the DNA sequence presented in FIG. 1 or upon allelic or homologous genomic and/or DNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention. In order to be sufficiently selective and potent for inhibition, such antisense oligonucleotides should comprise at least 7 (Wagner et al., Nature Biotechnology 14:840-844, 1996) and more preferably, at least 15 consecutive bases which are complementary to the target. Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases. Although oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N-terminal or 5′ upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3′-untranslated regions may be targeted. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs. In addition, the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol. Neurobiol. 14(5):439-457. 1994) and at which proteins are not expected to bind. Finally, although FIG. 1 discloses cDNA sequence, one of ordinary skill in the art may easily derive the genomic DNA corresponding to the cDNA of FIG. 1. Thus, the present invention also provides for antisense oligonucleotides which are complementary to the genomic DNA corresponding to FIG. 1. Similarly, antisense to allelic or homologous DNAs and genomic DNAs are enabled without undue experimentation.

[0019] In one set of embodiments, the antisense oligonucleotides of the invention may be composed of “natural” deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5′ end of one native nucleotide and the 3′ end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage. These oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors.

[0020] In preferred embodiments, however, the antisense oligonucleotides of the invention also may include “modified” oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness.

[0021] The term “modified oligonucleotide” as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic intemucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5′ end of one nucleotide and the 3′ end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide. Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, phosphate triesters, acetamidates, peptides, and carboxymethyl esters.

[0022] The term “modified oligonucleotide” also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3′ position and other than a phosphate group at the 5′ position. Thus modified oligonucleotides may include a 2′-O-alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. Modified oligonucleotides also can include base analogs such as C-5 propyne modified bases (Wagner et al., Nature Biotechnology 14:840-844, 1996). The present invention, thus, contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding PI3K-C2α proteins, together with pharmaceutically acceptable carriers.

[0023] Antisense oligonucleotides may be administered as part of a pharmaceutical composition. Such a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art. The compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term “physiologically acceptable” refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.

[0024] It follows from the foregoing description that it is an object of this invention to isolate the nucleic acid sequence and derived amino acid sequence of the first human class II PI3 kinase.

[0025] It is a further object of this invention to recombinantly manufacture human PI3K-C2α kinase.

[0026] It is a further object of this invention to use computer assisted three dimensional reconstruction of human PI3K-C2α kinase protein to facilitate the identification of agonists or antagonists of PI3K-C2α.

[0027] It is a yet further object to use said three dimensional reconstruction to design ligands that may modulate the biochemical activity of human PI3-C2α kinase.

[0028] It is yet a further object of this invention to manufacture an antibody, preferably monoclonal, to PI3K-C2α, and more preferably further still to manufacture a humanised antibody to PI3K-C2α.

[0029] It is yet a further object of this invention that the monoclonal antibody be specific to class II PI3 kinases.

[0030] It is yet a further object of this invention to provide an assay kit for use in identifying said protein.

[0031] According to the first aspect of the invention there is provided the isolated nucleic acid molecule and/or corresponding protein of human PI3K-C2α as shown in FIG. 1, including any deletions, additions or substitutions, or a complementary sequence thereto.

[0032] In a preferred embodiment of the invention said complementary sequence binds to said nucleic acid sequence under stringent conditions. Preferably, these conditions include 0.5M sodium phosphate, pH7.2, 7% SDS, 1 mM EDTA at 65° C.

[0033] In yet a further preferred embodiment of the invention said deletions, additions or substitutions do not alter the function of the protein or indeed do not alter the function in a deleterious manner thus, in one example, a nucleic acid molecule may be provided that binds to the relevant gene to prevent expression of PI3K-C2α.

[0034] In yet a further preferred embodiment of the invention said deletions, additions or substitutions do alter the function of the protein. Thus, in one example, a mutation in PI3K-C2α could produce a dominant negative mutation that results in the inactivation of endogenous PI3K-C2α in transfected or transformed cells.

[0035] According to a second aspect of the invention there is provided an antibody, or at least an effective part thereof, which binds at least with a selective part of said human PI3K-C2α. Ideally said effective part comprises FAb fragments. Ideally said antibody is polyclonal or monoclonal and ideally further still said antibody is specific to class II PI3 Kinases. Ideally said antibody is humanised by recombinant methods to contain the variable region of said antibody with an invariant or constant region of a human antibody.

[0036] Ideally said antibody is provided with a marker including a conventional label or tag, for example a radioactive and/or fluorescent and/or epitope label or tag.

[0037] More ideally still said humanized monoclonal antibody to class II PI3- kinases is produced as a fusion polypeptide in an expression vector suitably adapted for transfection or transformation of prokaryotic or eukaryotic cells.

[0038] According to a yet further aspect of the invention there is provided a construct including all, or part of, the said nucleic acid molecule of the invention.

[0039] In a preferred embodiment of the invention said construct is adapted to be a cloning vehicle for human PI3K-C2α.

[0040] In a preferred embodiment of the invention said cloning vehicle may be used for expression of the protein sequence of human PI3K-C2α in transformed or transfected prokaryotic and eukaryotic cells. Preferably said cloning vehicle is adapted for use in E.coli, mammalian, insect, amphibian or fungal cells.

[0041] In a preferred embodiment of the invention said construct includes nucleic acid encoding at least a part of human PI3K-C2α which is functionally linked to a promoter so as to provide for either constitutive or inducible expression of human PI3K-C2α kinase. Preferably further still said construct is adapted for cell/tissue specific expression of PI3K-C2α.

[0042] According to a yet further aspect of the invention there is provided cells transformed or transfected with the cloning vehicle of the invention and so including the nucleic acid sequence molecule encoding human PI3K-C2α or a part thereof.

[0043] According to a yet further aspect of the invention there is provided recombinantly manufactured human PI3K-C2α.

[0044] According to a yet further aspect of the invention there is provided human PI3K-C2α kinases or a fraction thereof, which is characterised by insensitivity to the PI3 kinase(s) inhibitor Wortmannin and/or LY294002.

[0045] In a further preferred embodiment of this aspect of the invention said protein is also, or alternatively, characterised by the absence of a p85 binding site.

[0046] In a further preferred embodiment of this aspect of the invention said protein is also, or alternatively, characterised by the absence of a type II polyproline motif present in Drosophilia P13K_(—)68D and cpk.

[0047] In a yet a further preferred embodiment of this aspect of the invention said protein is also, or alternatively, characterised by divergent amino terminal region when compared to other class II PI3 kinases.

[0048] In a yet a further preferred embodiment of this aspect of the invention said protein is characterised by a molecular weight of 190 Kda.

[0049] In yet a further preferred embodiment of this aspect of the invention said protein is also, or alternatively, characterised by an in vitro substrate specificity that is restricted to PtdIns, PtdIns(4)P or at least PtdIns, PtdIns(4)P.

[0050] In any of the above embodiments of the invention said fraction ideally comprises the full length amino acid sequence or truncations thereof.

[0051] According to a further aspect of the invention there is provided the isolated genomic DNA and/or cDNA of human PI3K-C2α kinase according to any previous aspect of the invention.

[0052] According to a yet further aspect of the invention there is provided a process for the isolation of human PI3K-C2α kinase which process includes a PCR reaction.

[0053] In a preferred process of the invention said PCR reaction comprises use of at least two primers adapted to bind to at least one selected part of nucleic acid of human PI3K-C2α; and further said process also comprises the provision of conditions for amplifying at least one selected part of said nucleic acid using said primers; and further, following amplification, said process comprises the purification of the amplified product(s); and, optionally, isolation of at least one amplified fragment.

[0054] In a preferred process of the invention said primers are designed to selectively bind to the nucleic acid of class II kinases and ideally class II human kinases.

[0055] In yet a further preferred embodiment of the invention said primers are as follows: sense: 5′GGNGA T/C GA T/C T/C T A/G CGNCA A/G GA3′ (SEQ ID NO:4) antisense: 5′A/G AA A/G TGICC A/G AA A/G TC A/G/T AT A/G TG A/G TG A/G AA3′ (SEQ ID NO:5)

[0056] In yet a further preferred embodiment of the invention said primers include any one or more deletions, additions or substitutions which do not deleteriously affect the functional effectiveness of the primers.

[0057] According to a yet further aspect of the invention there is provided the use of any one of more of the aforementioned primers to isolate class II PI3 kinases and ideally human PI3K-C2α.

[0058] According to a yet further aspect of the invention there is provided the use of any one or more of the aforementioned primers to isolate human PI3 kinases and ideally human PI3K-C2α.

[0059] According to a yet further aspect of the invention there is provided an agent which is adapted to interact with the amino terminal region, or any part thereof, of human PI3K-C2α kinase.

[0060] According to a yet further aspect of the invention there is provided an assay kit for identifying human PI3K-C2α either intracellularly or in a cellular extract.

[0061] In a preferred embodiment of the invention said assay kit is a diagnostic kit.

[0062] Preferably said assay kit, or said diagnostic kit makes use of any one or more of the following techniques: western blotting; immunoprecipitation; immunofluorescence on whole cells or tissue sections; or quantitative ELISA reactions.

[0063] According to a yet further aspect of the invention there is provided a method for identifying agonists or antagonists effective at enhancing or blocking the activity of the polypeptide of the invention which comprises screening candidate molecules for such activity using the polypeptide, or fragments thereof the invention.

[0064] Ideally, screening may involve artificial techniques such as computer-aided techniques or conventional laboratory techniques.

[0065] Alternatively, the method of the invention may involve competitive binding assays in order to identify agents that selectively and ideally irreversibly bind to the polypeptide of the invention.

[0066] According to a yet further aspect of the invention there is provided a pharmaceutical or veterinary composition comprising an agent effective at enhancing or blocking the activity of the polypeptide of the invention which has been formulated for pharmaceutical or veterinary use and which optionally also includes a dilutant, carrier or excipient and/or is in unit dosage form.

[0067] According to a yet further aspect of the invention there is provided antisense oligonucleotide or a modified antisense oligonucleotide as hereindescribed adapted to hybridise to at least part of the nucleic acid molecule shown in FIG. 1.

[0068] Thus, in general terms the invention describes the nucleic acid sequence and derived amino acid sequence of a novel human class II PI3 kinase, human PI3K-C2α, which has novel features in relation to sequence, biochemistry and responses to well characterised inhibitors of PI3 kinases.

BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The invention will be described in further detail with reference to illustrative preferred embodiments and experimental results depicted in the accompanying drawings in which:

[0070]FIG. 1 represents the complete cDNA sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO:2) of PI3K-C2α;

[0071]FIGS. 2a, 2 b and 2 c show a comparison of the conserved domains found in type II PI3 kinases and in particular the kinase domains and C2-domains;

[0072]FIG. 3 represents a northern blot the distribution of mRNA encoding PI3K-C2α in various tissues;

[0073]FIGS. 4a and 4 b represent the in vitro substrate specificity of PI3K-C2α in the presence of Mg²⁺ and Mn²⁺, p110α is included as a positive control;

[0074]FIG. 5 represents the resistance of recombinant PI3K-C2α to the PI3-kinase inhibitors Wortmannin and LY 294002, (p110α=•, recombinant PI3K-C2α=o);

[0075]FIG. 6 is a diagrammatic representation of the sub-cellular fractionation of Cos 7 cells into various membrane fractions;

[0076]FIG. 7 represents a western blot of cell lysates prepared from various cell-lines and probed with antisera to PI3K-C2α;

[0077]FIG. 8 represents a western blot of sub-cellular fractions prepared from Cos 7 cells and probed with antisera to PI3K-C2α; and

[0078]FIG. 9 represents an immunofluoresence image of Cos7 cells expressing recombinant glu-tagged PI3K-C2α.

MATERIALS AND METHODS Identification of PI3K-C2α cDNA

[0079] Poly A+RNA was isolated from U937 cells using oligo(dT) cellulose (Stratagene) and used to synthesise first strand cDNA with AMV reverse transcriptase (Pharmacia). PCR reactions were then performed using Taq DNA polymerase (Life Technologies), first strand cDNA, 4 mM Mg2+ and 1 μM primers. The nucleotide sequences of the primers used were sense—GGNGA T/C GA T/C T/C T A/G CGNCA A/G GA (SEQ ID NO:4) [GDDLRQD/E (SEQ ID NO:9)] and antisense—A/G AA A/G TGICC A/G AA A/G TC A/G/T AT A/G TG A/G TG A/G AA (SEQ ID NO:5) [FHIDFGHF (SEQ ID NO:10)]. To facilitate subsequent cloning, an EcoRI restriction site was incorporated into the 5′ nd of each primer. The conditions used were 30 cycles of 94° C. for 30s, 56° C. for 30s, and 72° C. for 30s. After sub cloning into a Bluescript-SK vector, individual clones were selected and sequenced (ABI).

Isolation of PI3K-C2α cDNA

[0080] The PCR fragment was excised from the vector, labelled with ³² P dCTP using random primers (Amersham) and used to screen a 1 λzap U937 cDNA library (Stratagene). Approximately 3×10⁶ plaques were plated and transferred onto Hybond-N filters (Amersham). These were hybridised with the probe for 16 hours at 65° C. in 0.5M sodium phosphate pH7.2, 7% SDS and 1 mM EDTA. After this time, the filters were washed twice with 0.5x SSC/0.1% SDS for 20 minutes at 60° C. and positive clones identified by autoradiography. After three rounds of screening, clones were plaque purified and the cDNA insert in Bluescript-SK was rescued using ExAssist helper phage (Stratagene). The nucleotide sequence of the longest clone was determined using both Taq di deoxy terminator cycle sequencing (ABI) and di deoxy chain termination using T7 DNA polymerase and a ³⁵S thio dATP (Lark Sequencing Technologies).

[0081] The 5′ end of the eDNA was obtained by rapid amplification of cDNA ends. cDNA was synthesised (Life Technologies) using primers complementary to the original cDNA sequence [CTCTTCCTCATGGTCTAATACCTCCAC (SEQ ID NO:3) and TATCTCCAAATCAGTCCTTGCTTTCCC (SEQ ID NO:6)] from poly A+ mRNA obtained from U937 cells. After sequencing, this fragment was ligated onto the parent cDNA in a pBK-CMV vector (Stratagene) to produce the full length PI3K-C2α cDNA.

Northern Blot Analysis

[0082] Multiple human tissue poly A+ mRNA blots (Clontech) were hybridised with a 1.46 kb cDNA fragment corresponding to nucleotides 789-2249 of the PI3K-C2α coding sequence (SEQ ID NO:1). The probe was labelled using [α ³²p] dCTP and random primers (Amersham). Hybridizations were performed in Express Hyb (Clontech) at 65° C. for 16 hours. The blots were washed twice with 2xSSC, 0.05%SDS at room temperature and twice in 1xSSC, 0.1%SDS at 50° C. Bands were visualised by autoradiography.

Plasmid Constructs and Expression of Recombinant Protein in Sf9 Cells

[0083] PI3K-C2α cDNA corresponding to amino acid residues 148-1686 of SEQ ID NO:2 was ligated in the pAcGex vector (44). Recombinant DNA (2 μg) was transfected into Sf9 cells with 0.25 μg BaculoGold DNA (Pharmingen) using Lipofectin (Gibco). Infections and amplifications (6×10⁵ cells/ml) of the viral stocks were performed for 48-60 hours or 96 hours respectively (45). After protein expression, cells were harvested and lysed in 10 mM Tris/HCl (pH7.6), 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100μ M Na₃VO₄, 1% Triton X-100 and 1 mM phenylmethysulphonyl fluoride (lysis buffer). The extracts were incubated with glutathione-Sepharose beads (Pharmacia) for 2-4 hours at 4° C. Bound proteins were recovered by centrifugation and after repeated washing with lysis buffer, the protein was transferred to storage buffer (50% ethyleneglycol, 5 mM EDTA, 40 mM Tris-HCl pH7.4, 5 mM dithiothriotol, 10 mM benzamidine) at −20° C.

I. Generation of Antisera to PI3K-C2α

[0084] An amino-terminal fragment of the PI3K-C2α cDNA (1-1011bp of SEQ ID NO: 1) was subcloned into pGex-2T vector. This plasmid was used to transform bacteria and the expression of a glutathione S-transferase (GST) fusion protein was induced. This protein was isolated using glutathione-sepharose beads and then eluted upon addition of excess glutathione. The GST fusion protein was used to immunise two rabbits, one of which produced antisera suitable for immunoprecipitation and western blotting PI3K-C2α from cell lysates.

II. Western blotting

[0085] Various cell lines were extracted with Laemelli sample buffer and fractionated by SDS-PAGE. The proteins were then transferred onto PVDF membrane which was probed with the anti- PI3K-C2α antisera. After washing, the blots were incubated with goat -anti -rabbit HRP antibody and proteins were visualised by ECL ( Amersham).

A. Subcellular Fractionation of PI3K-C2α

[0086] The subcellular distribution of endogenous PI3K-C2α protein was examined by differential centrifugation of cell homogenates. Cells were disrupted either by dounce homogenisation or 10 passages through a 25 gauge needle in Tris/sucrose buffer ( 0.25M sucrose, 20 mM Tris/HCl pH 7.4, 1 mM sodium orthovanadate, 1 mM PMSF, 10 μg/ml leupeptin, 200 KIU/ml aprotonin, 1 μg/ml antipain, 400 μg/ml benzamidine and 1 μg/ml pepstatin). The homogenate (homog) was centrifuged at 3000 g for 10 minutes to remove nuclei and unbroken cells. The supernatant (s/n) was spun at 18000 g to obtain the plasma membrane fraction (pm) and the resultant supernatant centrifuged for an additional 30 minutes at 350000 g. The pellet obtained was termed the low density microsomal fraction (LDM) and the supernatant cytosol (cyt). All manipulations were performed at 4° C. Fractions were extracted in Laemelli sample buffer, analyzed by SDS-PAGE and visualized by western blotting.

[0087] Monkey Kidney Cos cells were transiently transfected with a 5′ and 3′ glu tagged PI3K-C2α construct ligated in a pMT-SM vector. These cells were used 48 hours following electroporation. For fractionation studies, proteins were visualized by a anti- glu tag monoclonal antibody following western blotting. These cells were also used to examine the localization of the PI3K-C2α protein by confocal microscopy. For this purpose, electroporated cells were plated onto glass cover slips. After 48 hours, the cells were fixed, permeabilized and incubated with anti-glu tag monoclonal antibody for two hours. Cells were then washed and incubated with anti-mouse FITC coupled antibody. The staining pattern was then examined using a Zeiss confocal microscope.

Assay of PI 3-kinase Activity

[0088] Typically, PI 3-kinase assays were performed in a total volume of 50 μl containing 20 mM Hepes pH7.4. 100 mM NaCl, 0.1 mM EGTA, 0.1% β-mercaptoethanol, and 200 μM phosphoinositide. After preincubating sonicated lipid with sample for 10 minutes, the reactions were initiated upon the addition of 2.5 mM MgCl₂, 100 mM ATP (0.2μCiγ[³² P] ATP). Assays were incubated at 30° C. for 20 minutes and terminated with acidified chloroform:methanol. The extracted lipid products were analysed by thin layer chromatography using Silica gel 60 plates and chloroform:methanol:4M ammonium hydroxide (45:35:10) for assays examining phosphorylation of Ptdlns. When separations of PtdIns 3P, PtdIns (3,4) P₂ and PItdIns (3,4,5) P₃ was required, propan-1-ol: 2M acetic acid (65:35) was used. Phosphorylated lipids were visualised by either autoradiography or by phosphoimage analysis (Molecular Dynamics). Assays were linear with respect to time and enzyme addition.

Characterization of PI 3K-C2α Reaction Products by HPLC

[0089] Phosphorylation of PI was performed using recombinant protein as described above. The reaction products were extracted in acidified chloroform :methanol and deacylated with methylamine (46). HPLC analysis of glycerophosphoinositols was performed using a Partisphere SAX column (Whatman International) and eluted using a linear gradient of 1M (NH₄ )₂ HPO₄ (pH3.8) against water at 1 ml min⁻¹. Radioactive peaks were detected using an in-line detector (Reeve Analytical).

Results Cloning of PI3K-C2α

[0090] Degenerate primers and mRNA from the human cell line U937 were used to produce a number of partial cDNAs by RT-PCR. One product of these reactions was found to be a novel cDNA which was then used to screen a λ Zap cDNA library made from U937 cells. Twenty seven positive clones selected from 3×10⁶ plaques were obtained following tertiary screening. A 6.1 kb clone was sequenced and found to contain an open reading frame of 4618 nucleotides which could encode a putative catalytic domain. Since no definitive 5′ Kozak consensus sequence was found in this clone (24), RACE PCR was performed using cDNA from U937 cells. A 643 nucleotide extension to the original cDNA was generated. This cDNA fragment contained an ATG sequence which was in frame with the longest open reading frame of the parent clone and had an upstream stop codon. The RACE product was then ligated onto the parent clone to produce a composite cDNA of 5.1 kb which could encode a protein of 1686 amino acid residues that would have a calculated molecular mass of 190 kDa (FIG. 1).

[0091] The protein which was termed PI3K-C2α, has an amino acid sequence which when optimally aligned, displays an overall sequence identity of 32.5% with the sequences of drosophila PI3K_(—)68D and cpk (22,23) and 90.8% and 90.2% with those of the murine proteins mp170 (SEQ ID NO:7) (16) and mcpk (SEQ ID NO:8) (22), respectively (FIG. 2a). Two regions of amino acid sequence, HRI and HRII, define areas which have the greatest similarity to the sequences of other PI 3-kinases. The putative catalytic domain (HRl) is defined by residues 1137-1398 of the PI3K-C2α or α protein. Within this region, the amino acid sequence of PI3K-C2α is 62% identical to that of PI3K_(—)68D and cpk, 97% to mp170 and 99.6% to mcpk (FIG. 2c). When the HRl domain of PI3K-C2α is compared to that of p110α and PtdIns 3-kinase, sequence identity falls to only 45% and 35% respectively. Amino acid residues 871-1020 of PI3K-C2α define a region termed the PIK domain (HR2) which is also present in other lipid kinases as well as TOR2 (25). The function of this domain is currently unknown.

[0092] The amino acid sequence of the carboxy terminal region of PI3K-C2α (residues 1549-1686) shows strong sequence homology to a domain originally identified as a Ca²⁺ and phospholipid-binding module in protein kinase C, which is termed the C2 domain (13). In this region, the PI3K-C2α amino acid sequence is 96% identical to that of the C2 domains identified in cpk-m and p170, 39% identical to the C2 domain of PI3K_(—)68D and cpk and 32% and 24% identical to the C2A and C2B domains of synaptotagmin respectively (FIG. 2c). Within the amino terminal region, large insertions are necessary in the PI3K-C2α amino acid sequence to allow optimal alignment with that of Drosophila PI3K_(—)68D and cpk. It is perhaps significant that the type II polyproline motif PPLPPR identified in PI3K_(—)68D and cpk (residues 456-462) is absent in PI3K-C2α. Within this region however, numerous proline residues are evident. The amino terminus of PI3K-C2α lacks any clearly delineated regulatory domain.

[0093] Interestingly, alignment of the two murine amino acid sequences with that of PI3K-C2α shows that the p170 protein (SEQ ID NO:7) lacks the first 176 amino residues of the longer murine mcpk sequence (SEQ ID NO:8). The mcpk protein has a 28 amino acid residues deletion in this region (residues 275- 301 of the PI3K-C2α sequence) which is absent in both mp170 and PI3K-C2α(FIG. 2b). The significance of these sequence differences requires further investigation.

Tissue/Cell Expression of PI3K-C2α

[0094] Northern blot analysis carried out using poly A+RNA isolated from human tissue revealed that the cDNA encoding human PI3K-C2α hybridised to a 8 kb species in the RNA from a wide variety of tissues (FIG. 3). Highest levels of expression were found in the heart, placenta and ovary. The mRNA was only undetectable in the kidney. The size of the mRNA is consistent with the length of the full length cDNA. In testes, an additional smaller transcript of approximately 6.5 kb was also present and could have arisen from differential splicing.

[0095] Western blots of cell lysates from a variety of cell-lines using the anti-PI3K-C2α antisera confirms the presence of the 190 kDa polypeptide encoding PI3K-C2 in all cell-lines tested with the exception of the smooth muscle cell-line Rasmc, FIG. 7.

Characterisation of Lipid Substrate Specificity

[0096] To allow biochemical analysis of PI3K-C2α, a recombinant baculovirus was produced. The construct generated encoded a fusion protein which comprised the amino acid sequence of GST linked to residues 148-1686 of the PI3K-C2α protein. Sf9 cells were infected with the virus and after 2 days, the expressed protein was purified using glutathione-sepharose beads. The enzymatic activity and substrate specificity were investigated and compared to that of GST-p110α expressed in the same system. In the presence of Mg²⁺ p110α was able to utilise PtdIns, PtdIns(4)P₂ and PtdIns(4,5)P₂ as substrates to produce PtdIns3P, PtdIns(3,4) P₂ and PtdIns(3,4,5)P₃ respectively (FIG. 4a). In contrast, PI3K-C2α was able to phosphorylate PtdIns and PtdIns(4)P but could not use PtdIns(4,5)P₂. Like p110, the lipid kinase activity of PI3K-C2α displayed a cation dependence for Mg²⁺ and was found to be inactive in the presence of Mn²⁺. This ion dependence of the enzyme activity of PI3K-C2α contrasts with that of the human Ptd Ins 3-kinase which shows a preference the Mn²⁺ (14).

[0097] To confirm that PI3K-C2α, could phosphorylate PtdIns at the D-3 position on the inositol ring, anion exchange HPLC was performed upon the deacylated reaction products. A single peak of radioactive glycerolphosphate was obtained which co-eluted with the deacylated PtdIns 3-P produced by the action of recombinant p110α upon Ptd Ins (FIG. 4b). No additional products were observed confirming that PI3K-C2 specifically phosphorylates phosphatidylinositides at the D-3 position of their inositol ring.

Sensitivity to Inhibitors of PI 3-kinase Activity

[0098] The compounds Wortmannin and LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) have been used extensively to assess the involvement of PI 3-kinase in many different physiological processes. To date, all cloned human PI 3-kinases have been found to be sensitive to Wortmannin action at nanomolar concentrations. The effect of these inhibitors on the lipid kinase activity of PI3K-C2α was examined, using PtdIns as a substrate, in the presence of increasing concentrations of each inhibitor. In agreement with previous studies, p110 α activity was found to be sensitive to Wortmannin at low nM concentrations (lC₅₀=3 nM) with maximal attenuation of activity obtained at 100 nM (FIG. 5A). In contrast, the observed IC₅₀ using PI3K-C2α was 200 nM with maximal inhibition only obtained using Wortmannin at 10 μM. For LY294002, the IC₅₀ using p110α was 1.5 mM and maximal inhibition was obtained at 300 μM (FIG. 5B). Again PI3K-C2α was less sensitive than p110α to the action of the inhibitor (IC₅₀=20 μM). The lipid kinase activity of PI3K-C2 α was maximally attenuated using 2 mM LY294002.

III. Subcellular Localization of PI3K-C2α in Monkey Kidney Cos Cells

[0099] To determine the subcellular location of PI3K-C2α, Monkey Kidney Cos cells were subjected to physical disruption followed by fractionation by differential centrifugation, FIG. 6. FIG. 7 shows a western blot of various subcellular fractions prepared as described in materials and methods. A significant enrichment of PI3K-C2α polypeptide is shown in the LDM fraction when using the anti-amino terminus antisera, see FIG. 8A. This enrichment is also reflected, although to a lesser extent, when using cell fractions prepared from electroporated Monkey Kidney Cos cells expressing glu tagged PI3K-C2α, see FIG. 8B. The nature of the affinity of PI3K-C2α for microsomal membranes is currently unkown.

Discussion

[0100] In the present study the cloning and biochemical characterization of PI3K-C2α, a novel human PI 3-kinase of 190 kDa is described. Based on the presence of a C-terminal C2 domain and the phospholipid substrate specificity of PI3K-C2α, this enzyme like Drosophila PI3K_(—)68D (23), cpk (22) and the two murine PI 3-kinases mp170 (16) and mcpk (22) can be defined as a class II PI 3-kinase (26) (FIG. 2).

[0101] The amino acid sequence of PI3K-C2α is 90.8% and 90.2% identical to that of two recently cloned murine PI 3-kinases mp170 (16) and mcpk respectively (22). This level of sequence identity suggests that PI3K-C2α is the human homologue of a single mouse P13-kinase termed mp170 (27) or mcpk (28) which is encoded by the same gene. Despite the overall sequence identity between human PI3K-C2α and murine mp170 and mcpk, alignment of their N-termini shows that mp170 lacks the first 175 amino acid residues of both PI3K-C2α and cpk-m (FIG. 2). In this region mcpk lacks 28 amino acid residues which are present in both PI3K-C2α and mp170. Analysis of the two murine cDNAs suggests that these differences in the proteins may be the result of differential splicing of a single gene.

[0102] The putative catalytic domain (HRI) and the PIK domain (HR II) of PI3K-C2α are related to similar regions found in all Class I, II and III P13-kinases. However, PI3K-C2α appears to lack any clear consensus sequences which would delineate other functional domains. For example, PI3K-C2α lacks the amino terminal motif present in p110α, p110β and p110δ which mediates binding to p85 adaptors (29). Furthermore, PI3K-C2α does not contain the type II polyproline motif previously identified in P13K_(—b 68)D and cpk, although it does contain a large number of proline residues which might allow an interaction with SH3 domain-containing proteins through a non consensus binding motif (30).

[0103] The class II P13-kinases are in part characterized by their C-terminal C2 domain. C2 domains were originally identified in protein kinase C where they were shown to mediate Ca²⁺-dependent phospholipid binding (13). Subsequently, C2 domains, often occurring in tandem, have also been defined in a variety of other proteins including synaptotagmin, rabphillin 2A and cytosolic phospholipase A2 (31). In these proteins the C2 domain may confer similar biochemical properties. Functional heterogeneity has however, been observed amongst isolated C2 domains despite their high degree of sequence similarity. In addition to phospholipid binding, Ca2+ stimulates an association between synaptotagmin and syntaxin through the first C2A domain (32). In contrast, the second C2B domain is inactive in these assays suggesting that its function is not regulated by intracellular Ca²⁺ levels. Indeed, the second C2 domain of synaptotagmin was shown to display a Ca²⁺ independent binding to clathrin AP2 and polyinositol phosphates (33, 34). Recent studies have since demonstrated however, that the second C2 domain of synaptotagmin can also respond in a Ca²⁺ triggered manner mediating a dimerization event (35, 36). Studies using a GST-fusion protein of the PI3 K_(—)68D C2 domain demonstrated that, like the second C2 domain of synaptotagmin, it was only able to bind acidic phospholipids in a cation independent manner (23). It will be of interest to determine if the biochemical properties of the PI3K-C2α domain are more similar to the first or the second synaptotagmin C2 domain.

[0104] The biochemical analysis described here has shown that in vitro PI3K-C2α can phosphorylate both Ptd Ins and Ptd Ins (4)P in the D3 position of the inositol ring (FIG. 4). These findings are consistent with the enzyme having a lipid specificity similar to that of P13K₁₃ 68D and cpk (23), (22). The substrate specificity of the mouse homologue has only been reported by Virbasius et al who concluded that Ptd Ins was the major lipid substrate of mp170 in vitro since they found that PtdIns(4)P was only very poorly phosphorylated by their enzyme (16). These results are somewhat surprising considering the degree of sequence identity between the catalytic domains of PI3K-C2α and mpl70 (FIG. 2). One explanation for this difference could be that Virbasius et al used a crude brain lipid extract substrate in contrast to the purified lipid preparations we have used in the analysis of substrate specificity (16). Although it could be argued that use of the crude extract may be more physiological, it does not allow for an accurate quantitative comparison of the lipids present in the two preparations. Under these circumstances, a comparative assessment of the efficiency with which an enzyme could utilize a particular lipid substrate cannot be made accurately. Furthermore, a crude lipid preparation may contain factors which influence the lipid kinase activity of the recombinant protein.

[0105] The involvement of PI3-kinases in various processes of cell physiology has relied more and more on the use of the inhibitors Wortmannin and LY294002. Although initially described as an inhibitor of myosin light chain kinase at micromolar concentrations (37), Wortmannin was later shown to potently inhibit PDGF receptor-associated PI 3-kinase activity at 100 fold lower concentrations (2-5 nanoinolar). Used in the low nanomolar range, Wortmannin was shown to block the generation of 3-phosphorylated lipids both in vitro and in whole cells such as murine fibroblasts (38). On the basis of such findings, Wortmannin began to be used extensively as a specific inhibitor of PI 3-kinase activity thereby implicating the formation of 3-phosphorylated lipids in a number of physiological responses. When the effect of Wortmannin on the lipid kinase activity of recombinant PI3K-C2α was examined we found that the concentrations of Wortmannin required to inhibit PI3K-C2α activity were more similar to those previously used to inhibit myosin light chain kinase (IC₅₀=170-200 nM) (37, 39). Similarly, data presented using the mp170 enzyme suggests that it is also less sensitive to Wortmannin treatment than p110α (16). LY294002 is a structurally unrelated inhibitor of PI 3-kinase activity and PI3K-C2α is also less sensitive than p110α to this inhibitor. The difference in sensitivity between PI3K-C2α and p110α is however, only approximately one order of magnitude. The relevance of this differential sensitivity is unclear. Studies using p110α have shown that Wortmannin inactivates kinase activity by a covalent modification of Lys-802, a residue involved in the phosphate transfer reaction (40). This residue is present in all PI 3-kinase family members including PI3K-C2α. Further molecular modelling and structural studies may elucidate the nature of this Wortmannin resistance. These results with PI3K-C2α contrast not only with those derived from studies of Class I and III human PI 3-kinases but also to those obtained with the Drosophila PI3K_(—)68D which displays a sensitivity to Wortmannin which is similar to that of p110α (23). The concentrations of both Wortmannin and LY294002 required to abolish PI3K-C2α activity are therefore likely to have precluded its inhibition in many experiments where PI 3-kinase activity has been studied. These findings may thus require many investigators to re-evaluate a possible involvement of PI3-kinase action in their physiological studies. Questions relating to the precise role PI3K-C2α plays in cells will consequently be hampered by the absence of suitable inhibitors of its lipid kinase activity.

[0106] The foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting. Since modifications of the described embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed broadly to include all variations falling within the scope of the appended claims and equivalents thereof.

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1 11 1 5061 DNA Homo sapiens CDS (1)..(5058) 1 atg gct cag ata ttt agc aac agc gga ttt aaa gaa tgt cca ttt tca 48 Met Ala Gln Ile Phe Ser Asn Ser Gly Phe Lys Glu Cys Pro Phe Ser 1 5 10 15 cat ccg gaa cca aca aga gca aaa gat gtg gac aaa gaa gaa gca tta 96 His Pro Glu Pro Thr Arg Ala Lys Asp Val Asp Lys Glu Glu Ala Leu 20 25 30 cag atg gaa gca gag gct tta gca aaa ctg caa aag gat aga caa gtg 144 Gln Met Glu Ala Glu Ala Leu Ala Lys Leu Gln Lys Asp Arg Gln Val 35 40 45 act gac aat cag aga ggc ttt gag ttg tca agc agc acc aga aaa aaa 192 Thr Asp Asn Gln Arg Gly Phe Glu Leu Ser Ser Ser Thr Arg Lys Lys 50 55 60 gca cag gtt tat aac aag cag gat tat gat ctc atg gtg ttt cct gaa 240 Ala Gln Val Tyr Asn Lys Gln Asp Tyr Asp Leu Met Val Phe Pro Glu 65 70 75 80 tca gat tcc caa aaa aga gca tta gat att gat gta gaa aag ctc acc 288 Ser Asp Ser Gln Lys Arg Ala Leu Asp Ile Asp Val Glu Lys Leu Thr 85 90 95 caa gct gaa ctt gag aaa cta ttg ctg gat gac agt ttc gag act aaa 336 Gln Ala Glu Leu Glu Lys Leu Leu Leu Asp Asp Ser Phe Glu Thr Lys 100 105 110 aaa aca cct gta tta cca gtt act cct att ctg agc cct tcc ttt tca 384 Lys Thr Pro Val Leu Pro Val Thr Pro Ile Leu Ser Pro Ser Phe Ser 115 120 125 gca cag ctc tat ttt aga cct act att cag aga gga cag tgg cca cct 432 Ala Gln Leu Tyr Phe Arg Pro Thr Ile Gln Arg Gly Gln Trp Pro Pro 130 135 140 gga tta cct ggg cct tcc act tat gct tta cct tct att tat cct tct 480 Gly Leu Pro Gly Pro Ser Thr Tyr Ala Leu Pro Ser Ile Tyr Pro Ser 145 150 155 160 act tac agt aaa cag gct gca ttc caa aat ggc ttc aat cca aga atg 528 Thr Tyr Ser Lys Gln Ala Ala Phe Gln Asn Gly Phe Asn Pro Arg Met 165 170 175 ccc act ttt cca tct aca gaa cct ata tat tta agt ctt ccg gga caa 576 Pro Thr Phe Pro Ser Thr Glu Pro Ile Tyr Leu Ser Leu Pro Gly Gln 180 185 190 tct cca tat ttc tca tat cct ttg aca cct gcc aca ccc ttt cat cca 624 Ser Pro Tyr Phe Ser Tyr Pro Leu Thr Pro Ala Thr Pro Phe His Pro 195 200 205 caa gga agc tta cct atc tat cgt cca gta gtc agt act gac atg gca 672 Gln Gly Ser Leu Pro Ile Tyr Arg Pro Val Val Ser Thr Asp Met Ala 210 215 220 aaa cta ttt gac aaa ata gct agt aca tca gaa ttt tta aaa aat ggg 720 Lys Leu Phe Asp Lys Ile Ala Ser Thr Ser Glu Phe Leu Lys Asn Gly 225 230 235 240 aaa gca agg act gat ttg gag ata aca gat tca aaa gtc agc aat cta 768 Lys Ala Arg Thr Asp Leu Glu Ile Thr Asp Ser Lys Val Ser Asn Leu 245 250 255 cag gta tct cca aag tct gag gat atc agt aaa ttt gac tgg tta gac 816 Gln Val Ser Pro Lys Ser Glu Asp Ile Ser Lys Phe Asp Trp Leu Asp 260 265 270 ttg gat cct cta agt aag cct aag gtg gat aat gtg gag gta tta gac 864 Leu Asp Pro Leu Ser Lys Pro Lys Val Asp Asn Val Glu Val Leu Asp 275 280 285 cat gag gaa gag aaa aat gtt tca agt ttg cta gca aag gat cct tgg 912 His Glu Glu Glu Lys Asn Val Ser Ser Leu Leu Ala Lys Asp Pro Trp 290 295 300 gat gct gtt ctt ctt gaa gag aga tcg aca gca aat tgt cat ctt gaa 960 Asp Ala Val Leu Leu Glu Glu Arg Ser Thr Ala Asn Cys His Leu Glu 305 310 315 320 aga aag gtg aat gga aaa tcc ctt tct gtg gca act gtt aca aga agc 1008 Arg Lys Val Asn Gly Lys Ser Leu Ser Val Ala Thr Val Thr Arg Ser 325 330 335 cag tct tta aat att cga aca act cag ctt gca aaa gcc cag ggc cat 1056 Gln Ser Leu Asn Ile Arg Thr Thr Gln Leu Ala Lys Ala Gln Gly His 340 345 350 ata tct cag aaa gac cca aat ggg acc agt agt ttg cca act gga agt 1104 Ile Ser Gln Lys Asp Pro Asn Gly Thr Ser Ser Leu Pro Thr Gly Ser 355 360 365 tct ctt ctt caa gaa gtt gaa gta cag aat gag gag atg gca gct ttt 1152 Ser Leu Leu Gln Glu Val Glu Val Gln Asn Glu Glu Met Ala Ala Phe 370 375 380 tgt cga tcc att aca aaa ttg aag acc aaa ttt cca tat acc aat cac 1200 Cys Arg Ser Ile Thr Lys Leu Lys Thr Lys Phe Pro Tyr Thr Asn His 385 390 395 400 cgc aca aac cca ggc tat ttg tta agt cca gtc aca gcg caa aga aac 1248 Arg Thr Asn Pro Gly Tyr Leu Leu Ser Pro Val Thr Ala Gln Arg Asn 405 410 415 ata tgc gga gaa aat gct agt gtg aag gtc tcc att gac att gaa gga 1296 Ile Cys Gly Glu Asn Ala Ser Val Lys Val Ser Ile Asp Ile Glu Gly 420 425 430 ttt cag cta cca gtt act ttt acg tgt gat gtg agt tct act gta gaa 1344 Phe Gln Leu Pro Val Thr Phe Thr Cys Asp Val Ser Ser Thr Val Glu 435 440 445 atc att ata atg caa gcc ctt tgc tgg gta cat gat gac ttg aat caa 1392 Ile Ile Ile Met Gln Ala Leu Cys Trp Val His Asp Asp Leu Asn Gln 450 455 460 gta gat gtt ggc agc tat gtt cta aaa gtt tgt ggt caa gag gaa gtg 1440 Val Asp Val Gly Ser Tyr Val Leu Lys Val Cys Gly Gln Glu Glu Val 465 470 475 480 ctg cag aat aat cat tgc ctt gga agt cat gag cat att caa aac tgt 1488 Leu Gln Asn Asn His Cys Leu Gly Ser His Glu His Ile Gln Asn Cys 485 490 495 cga aaa tgg gac aca gaa att aga cta caa ctc ttg acc ttc agt gca 1536 Arg Lys Trp Asp Thr Glu Ile Arg Leu Gln Leu Leu Thr Phe Ser Ala 500 505 510 atg tgt caa aat ctg gcc cga aca gca gaa gat gat gaa aca ccc gtg 1584 Met Cys Gln Asn Leu Ala Arg Thr Ala Glu Asp Asp Glu Thr Pro Val 515 520 525 gat tta aac aaa cac ctg tat caa ata gaa aaa cct tgc aaa gaa gcc 1632 Asp Leu Asn Lys His Leu Tyr Gln Ile Glu Lys Pro Cys Lys Glu Ala 530 535 540 atg acg aga cac cct gtt gaa gaa ctc tta gat tct tat cac aac caa 1680 Met Thr Arg His Pro Val Glu Glu Leu Leu Asp Ser Tyr His Asn Gln 545 550 555 560 gta gaa ctg gct ctt caa att gaa aac caa cac cga gca gta gat caa 1728 Val Glu Leu Ala Leu Gln Ile Glu Asn Gln His Arg Ala Val Asp Gln 565 570 575 gta att aaa gct gta aga aaa atc tgt agt gct tta gat ggt gtc gag 1776 Val Ile Lys Ala Val Arg Lys Ile Cys Ser Ala Leu Asp Gly Val Glu 580 585 590 act ctt gcc att aca gaa tca gta aag aag cta aag aga gca gtt aat 1824 Thr Leu Ala Ile Thr Glu Ser Val Lys Lys Leu Lys Arg Ala Val Asn 595 600 605 ctt cca agg agt aaa act gct gat gtg act tct ttg ttt gga gga gaa 1872 Leu Pro Arg Ser Lys Thr Ala Asp Val Thr Ser Leu Phe Gly Gly Glu 610 615 620 gac act agc agg agt tca act agg ggc tca ctt aat cct gaa aat cct 1920 Asp Thr Ser Arg Ser Ser Thr Arg Gly Ser Leu Asn Pro Glu Asn Pro 625 630 635 640 gtt caa gta agc ata aac caa tta act gca gca att tat gat ctt ctc 1968 Val Gln Val Ser Ile Asn Gln Leu Thr Ala Ala Ile Tyr Asp Leu Leu 645 650 655 aga ctc cat gca aat tct ggt agg agt cct aca gac tgt gcc caa agt 2016 Arg Leu His Ala Asn Ser Gly Arg Ser Pro Thr Asp Cys Ala Gln Ser 660 665 670 agc aag agt gtc aag gaa gca tgg act aca aca gag cag ctc cag ttt 2064 Ser Lys Ser Val Lys Glu Ala Trp Thr Thr Thr Glu Gln Leu Gln Phe 675 680 685 act att ttt gct gct cat gga att tca agt aat tgg gta tca aat tat 2112 Thr Ile Phe Ala Ala His Gly Ile Ser Ser Asn Trp Val Ser Asn Tyr 690 695 700 gaa aaa tac tac ttg ata tgt tca ctg tct cac aat gga aag gat ctt 2160 Glu Lys Tyr Tyr Leu Ile Cys Ser Leu Ser His Asn Gly Lys Asp Leu 705 710 715 720 ttt aaa cct att caa tca aag aag gtt ggc act tac aag aat ttc ttc 2208 Phe Lys Pro Ile Gln Ser Lys Lys Val Gly Thr Tyr Lys Asn Phe Phe 725 730 735 tat ctt att aaa tgg gat gaa cta atc att ttt cct atc cag ata tca 2256 Tyr Leu Ile Lys Trp Asp Glu Leu Ile Ile Phe Pro Ile Gln Ile Ser 740 745 750 caa ttg cca tta gaa tca gtt ctt cac ctt act ctt ttt gga att tta 2304 Gln Leu Pro Leu Glu Ser Val Leu His Leu Thr Leu Phe Gly Ile Leu 755 760 765 aat cag agc agt gga agt tcc cct gat tct aat aag cag aga aag gga 2352 Asn Gln Ser Ser Gly Ser Ser Pro Asp Ser Asn Lys Gln Arg Lys Gly 770 775 780 cca gaa gct ttg ggc aaa gtt tct tta cct ctt tgt gac ttt aga cgg 2400 Pro Glu Ala Leu Gly Lys Val Ser Leu Pro Leu Cys Asp Phe Arg Arg 785 790 795 800 ttt tta aca tgt gga act aaa ctt cta tat ctt tgg act tca tca cat 2448 Phe Leu Thr Cys Gly Thr Lys Leu Leu Tyr Leu Trp Thr Ser Ser His 805 810 815 aca aat tct gtt cct gga aca gtt acc aaa aaa gga tat gtc atg gaa 2496 Thr Asn Ser Val Pro Gly Thr Val Thr Lys Lys Gly Tyr Val Met Glu 820 825 830 aga ata gtg cta cag gtt gat ttt cct tct cct gca ttt gat att att 2544 Arg Ile Val Leu Gln Val Asp Phe Pro Ser Pro Ala Phe Asp Ile Ile 835 840 845 tat aca act cct caa gtt gac aga agc att ata cag caa cat aac tta 2592 Tyr Thr Thr Pro Gln Val Asp Arg Ser Ile Ile Gln Gln His Asn Leu 850 855 860 gaa aca cta gag aat gat ata aaa ggg aaa ctt ctt gat att ctt cat 2640 Glu Thr Leu Glu Asn Asp Ile Lys Gly Lys Leu Leu Asp Ile Leu His 865 870 875 880 aaa gac tca tca ctt gga ctt tct aaa gaa gat aaa gct ttt tta tgg 2688 Lys Asp Ser Ser Leu Gly Leu Ser Lys Glu Asp Lys Ala Phe Leu Trp 885 890 895 gag aaa cgt tat tat tgc ttc aaa cac cca aat tgt ctt cct aaa ata 2736 Glu Lys Arg Tyr Tyr Cys Phe Lys His Pro Asn Cys Leu Pro Lys Ile 900 905 910 tta gca agc gcc cca aac tgg aaa tgg ggt aat ctt gcc aaa act tac 2784 Leu Ala Ser Ala Pro Asn Trp Lys Trp Gly Asn Leu Ala Lys Thr Tyr 915 920 925 tca ttg ctt cac cag tgg cct gca ttg tac cca cta att gca ttg gaa 2832 Ser Leu Leu His Gln Trp Pro Ala Leu Tyr Pro Leu Ile Ala Leu Glu 930 935 940 ctt ctt gat tca aaa ttt gct gat cag gaa gta aga tcc cta gct gtg 2880 Leu Leu Asp Ser Lys Phe Ala Asp Gln Glu Val Arg Ser Leu Ala Val 945 950 955 960 acc tgg att gag gcc att agt gat gat gag cta aca gat ctt ctt cca 2928 Thr Trp Ile Glu Ala Ile Ser Asp Asp Glu Leu Thr Asp Leu Leu Pro 965 970 975 cag ttt gta caa gct ttg aaa tat gaa att tac ttg aat agt tca tta 2976 Gln Phe Val Gln Ala Leu Lys Tyr Glu Ile Tyr Leu Asn Ser Ser Leu 980 985 990 gtg caa ttc ctt ttg tcc agg gca ttg gga aat atc cag ata gca cac 3024 Val Gln Phe Leu Leu Ser Arg Ala Leu Gly Asn Ile Gln Ile Ala His 995 1000 1005 aat tta tat tgg ctt ctc aaa gat gcc ctg cat gat gta cag ttt 3069 Asn Leu Tyr Trp Leu Leu Lys Asp Ala Leu His Asp Val Gln Phe 1010 1015 1020 agt acc cga tac gaa cat gtt ttg ggt gct ctc ctg tca gta gga 3114 Ser Thr Arg Tyr Glu His Val Leu Gly Ala Leu Leu Ser Val Gly 1025 1030 1035 gga aaa cga ctt aga gaa gaa ctt cta aaa cag acg aaa ctt gta 3159 Gly Lys Arg Leu Arg Glu Glu Leu Leu Lys Gln Thr Lys Leu Val 1040 1045 1050 cag ctt tta gga gga gta gca gaa aaa gta agg cag gct agt gga 3204 Gln Leu Leu Gly Gly Val Ala Glu Lys Val Arg Gln Ala Ser Gly 1055 1060 1065 tca gcc aga cag gtt gtt ctc caa aga agt atg gaa cga gta cag 3249 Ser Ala Arg Gln Val Val Leu Gln Arg Ser Met Glu Arg Val Gln 1070 1075 1080 tcc ttt ttt cag aaa aat aaa tgc cgt ctc cct ctc aag cca agt 3294 Ser Phe Phe Gln Lys Asn Lys Cys Arg Leu Pro Leu Lys Pro Ser 1085 1090 1095 cta gtg gca aaa gaa tta aat att aag tcg tgt tcc ttc ttc agt 3339 Leu Val Ala Lys Glu Leu Asn Ile Lys Ser Cys Ser Phe Phe Ser 1100 1105 1110 tct aat gct gtc ccc cta aaa gtc aca atg gtg aat gct gac cct 3384 Ser Asn Ala Val Pro Leu Lys Val Thr Met Val Asn Ala Asp Pro 1115 1120 1125 ctg gga gaa gaa att aat gtc atg ttt aag gtt ggt gaa gat ctt 3429 Leu Gly Glu Glu Ile Asn Val Met Phe Lys Val Gly Glu Asp Leu 1130 1135 1140 cgg caa gat atg tta gct tta cag atg ata aag att atg gat aag 3474 Arg Gln Asp Met Leu Ala Leu Gln Met Ile Lys Ile Met Asp Lys 1145 1150 1155 atc tgg ctt aaa gaa gga cta gat ctg agg atg gta att ttc aaa 3519 Ile Trp Leu Lys Glu Gly Leu Asp Leu Arg Met Val Ile Phe Lys 1160 1165 1170 tgt ctc tca act ggc aga gat cga ggc atg gtg gag ctg gtt cct 3564 Cys Leu Ser Thr Gly Arg Asp Arg Gly Met Val Glu Leu Val Pro 1175 1180 1185 gct tcc gat acc ctc agg aaa atc caa gtg gaa tat ggt gtg aca 3609 Ala Ser Asp Thr Leu Arg Lys Ile Gln Val Glu Tyr Gly Val Thr 1190 1195 1200 gga tcc ttt aaa gat aaa cca ctt gca gag tgg cta agg aaa tac 3654 Gly Ser Phe Lys Asp Lys Pro Leu Ala Glu Trp Leu Arg Lys Tyr 1205 1210 1215 aat ccc tct gaa gaa gaa tat gaa aag gct tca gag aac ttt atc 3699 Asn Pro Ser Glu Glu Glu Tyr Glu Lys Ala Ser Glu Asn Phe Ile 1220 1225 1230 tat tcc tgt gct gga tgc tgt gta gcc acc tat gtt tta ggc atc 3744 Tyr Ser Cys Ala Gly Cys Cys Val Ala Thr Tyr Val Leu Gly Ile 1235 1240 1245 tgt gat cga cac aat gac aat ata atg ctt cga agc acg gga cac 3789 Cys Asp Arg His Asn Asp Asn Ile Met Leu Arg Ser Thr Gly His 1250 1255 1260 atg ttt cac att gac ttt gga aag ttt ttg gga cat gca cag atg 3834 Met Phe His Ile Asp Phe Gly Lys Phe Leu Gly His Ala Gln Met 1265 1270 1275 ttt ggc agc ttc aaa agg gat cgg gct cct ttt gtg ctg acc tct 3879 Phe Gly Ser Phe Lys Arg Asp Arg Ala Pro Phe Val Leu Thr Ser 1280 1285 1290 gat atg gca tat gtc att aat ggg ggt gaa aag ccc acc att cgt 3924 Asp Met Ala Tyr Val Ile Asn Gly Gly Glu Lys Pro Thr Ile Arg 1295 1300 1305 ttt cag ttg ttt gtg gac ctc tgc tgt cag gcc tac aac ttg ata 3969 Phe Gln Leu Phe Val Asp Leu Cys Cys Gln Ala Tyr Asn Leu Ile 1310 1315 1320 aga aag cag aca aac ctt ttt ctt aac ctc ctt tca ctg atg att 4014 Arg Lys Gln Thr Asn Leu Phe Leu Asn Leu Leu Ser Leu Met Ile 1325 1330 1335 cct tca ggg tta cca gaa ctt aca agt att caa gat ttg aaa tac 4059 Pro Ser Gly Leu Pro Glu Leu Thr Ser Ile Gln Asp Leu Lys Tyr 1340 1345 1350 gtt aga gat gca ctt caa ccc caa act aca gac gca gaa gct aca 4104 Val Arg Asp Ala Leu Gln Pro Gln Thr Thr Asp Ala Glu Ala Thr 1355 1360 1365 att ttc ttt act agg ctt att gaa tca agt ttg gga agc att gcc 4149 Ile Phe Phe Thr Arg Leu Ile Glu Ser Ser Leu Gly Ser Ile Ala 1370 1375 1380 aca aag ttt aac ttc ttc att cac aac ctt gct cag ctt cgt ttt 4194 Thr Lys Phe Asn Phe Phe Ile His Asn Leu Ala Gln Leu Arg Phe 1385 1390 1395 tct ggt ctt cct tct aat gat gag ccc atc ctt tca ttt tca cct 4239 Ser Gly Leu Pro Ser Asn Asp Glu Pro Ile Leu Ser Phe Ser Pro 1400 1405 1410 aaa aca tac tcc ttt aga caa gat ggt cga atc aag gaa gtc tct 4284 Lys Thr Tyr Ser Phe Arg Gln Asp Gly Arg Ile Lys Glu Val Ser 1415 1420 1425 gtt ttt aca tat cat aag aaa tac aac cca gat aaa cat tat att 4329 Val Phe Thr Tyr His Lys Lys Tyr Asn Pro Asp Lys His Tyr Ile 1430 1435 1440 tat gta gtc cga att ttg tgg gaa gga cag att gaa cca tca ttt 4374 Tyr Val Val Arg Ile Leu Trp Glu Gly Gln Ile Glu Pro Ser Phe 1445 1450 1455 gtc ttc cga aca ttt gtc gaa ttt cag gaa ctt cac aat aag ctc 4419 Val Phe Arg Thr Phe Val Glu Phe Gln Glu Leu His Asn Lys Leu 1460 1465 1470 agt att att ttt cca ctt tgg aag tta cca ggc ttt cct aat agg 4464 Ser Ile Ile Phe Pro Leu Trp Lys Leu Pro Gly Phe Pro Asn Arg 1475 1480 1485 atg gtt cta gga aga aca cac ata aaa gat gta gca gcc aaa agg 4509 Met Val Leu Gly Arg Thr His Ile Lys Asp Val Ala Ala Lys Arg 1490 1495 1500 aaa att gag tta aac agt tac tta cag agt ttg atg aat gct tca 4554 Lys Ile Glu Leu Asn Ser Tyr Leu Gln Ser Leu Met Asn Ala Ser 1505 1510 1515 acg gat gta gca gag tgt gat ctt gtt tgt act ttc ttc cac cct 4599 Thr Asp Val Ala Glu Cys Asp Leu Val Cys Thr Phe Phe His Pro 1520 1525 1530 tta ctt cgt gat gag aaa gct gaa ggg ata gct agg tct gca gat 4644 Leu Leu Arg Asp Glu Lys Ala Glu Gly Ile Ala Arg Ser Ala Asp 1535 1540 1545 gca ggt tcc ttc agt cct act cca ggc caa ata gga gga gct gtg 4689 Ala Gly Ser Phe Ser Pro Thr Pro Gly Gln Ile Gly Gly Ala Val 1550 1555 1560 aaa tta tcc atc tct tac cga aat ggt act ctt ttc atc atg gtg 4734 Lys Leu Ser Ile Ser Tyr Arg Asn Gly Thr Leu Phe Ile Met Val 1565 1570 1575 atg cat atc aaa gat ctt gtt act gaa gat gga gct gac cca aat 4779 Met His Ile Lys Asp Leu Val Thr Glu Asp Gly Ala Asp Pro Asn 1580 1585 1590 cca tat gtc aaa aca tac cta ctt cca gat aac cac aaa aca tcc 4824 Pro Tyr Val Lys Thr Tyr Leu Leu Pro Asp Asn His Lys Thr Ser 1595 1600 1605 aaa cgt aaa acc aaa att tca cga aaa acg agg aat ccg aca ttc 4869 Lys Arg Lys Thr Lys Ile Ser Arg Lys Thr Arg Asn Pro Thr Phe 1610 1615 1620 aat gaa atg ctt gta tac agt gga tat agc aaa gaa acc cta aga 4914 Asn Glu Met Leu Val Tyr Ser Gly Tyr Ser Lys Glu Thr Leu Arg 1625 1630 1635 cag cga gaa ctt caa cta agt gta ctc agt gca gaa tct ctg cgg 4959 Gln Arg Glu Leu Gln Leu Ser Val Leu Ser Ala Glu Ser Leu Arg 1640 1645 1650 gag aat ttt ttc ttg ggt gga gta acc ctg cct ttg aaa gat ttc 5004 Glu Asn Phe Phe Leu Gly Gly Val Thr Leu Pro Leu Lys Asp Phe 1655 1660 1665 aac ttg agc aaa gag acg gtt aaa tgg tat cag ctg act gcg gca 5049 Asn Leu Ser Lys Glu Thr Val Lys Trp Tyr Gln Leu Thr Ala Ala 1670 1675 1680 aca tac ttg taa 5061 Thr Tyr Leu 1685 2 1686 PRT Homo sapiens 2 Met Ala Gln Ile Phe Ser Asn Ser Gly Phe Lys Glu Cys Pro Phe Ser 1 5 10 15 His Pro Glu Pro Thr Arg Ala Lys Asp Val Asp Lys Glu Glu Ala Leu 20 25 30 Gln Met Glu Ala Glu Ala Leu Ala Lys Leu Gln Lys Asp Arg Gln Val 35 40 45 Thr Asp Asn Gln Arg Gly Phe Glu Leu Ser Ser Ser Thr Arg Lys Lys 50 55 60 Ala Gln Val Tyr Asn Lys Gln Asp Tyr Asp Leu Met Val Phe Pro Glu 65 70 75 80 Ser Asp Ser Gln Lys Arg Ala Leu Asp Ile Asp Val Glu Lys Leu Thr 85 90 95 Gln Ala Glu Leu Glu Lys Leu Leu Leu Asp Asp Ser Phe Glu Thr Lys 100 105 110 Lys Thr Pro Val Leu Pro Val Thr Pro Ile Leu Ser Pro Ser Phe Ser 115 120 125 Ala Gln Leu Tyr Phe Arg Pro Thr Ile Gln Arg Gly Gln Trp Pro Pro 130 135 140 Gly Leu Pro Gly Pro Ser Thr Tyr Ala Leu Pro Ser Ile Tyr Pro Ser 145 150 155 160 Thr Tyr Ser Lys Gln Ala Ala Phe Gln Asn Gly Phe Asn Pro Arg Met 165 170 175 Pro Thr Phe Pro Ser Thr Glu Pro Ile Tyr Leu Ser Leu Pro Gly Gln 180 185 190 Ser Pro Tyr Phe Ser Tyr Pro Leu Thr Pro Ala Thr Pro Phe His Pro 195 200 205 Gln Gly Ser Leu Pro Ile Tyr Arg Pro Val Val Ser Thr Asp Met Ala 210 215 220 Lys Leu Phe Asp Lys Ile Ala Ser Thr Ser Glu Phe Leu Lys Asn Gly 225 230 235 240 Lys Ala Arg Thr Asp Leu Glu Ile Thr Asp Ser Lys Val Ser Asn Leu 245 250 255 Gln Val Ser Pro Lys Ser Glu Asp Ile Ser Lys Phe Asp Trp Leu Asp 260 265 270 Leu Asp Pro Leu Ser Lys Pro Lys Val Asp Asn Val Glu Val Leu Asp 275 280 285 His Glu Glu Glu Lys Asn Val Ser Ser Leu Leu Ala Lys Asp Pro Trp 290 295 300 Asp Ala Val Leu Leu Glu Glu Arg Ser Thr Ala Asn Cys His Leu Glu 305 310 315 320 Arg Lys Val Asn Gly Lys Ser Leu Ser Val Ala Thr Val Thr Arg Ser 325 330 335 Gln Ser Leu Asn Ile Arg Thr Thr Gln Leu Ala Lys Ala Gln Gly His 340 345 350 Ile Ser Gln Lys Asp Pro Asn Gly Thr Ser Ser Leu Pro Thr Gly Ser 355 360 365 Ser Leu Leu Gln Glu Val Glu Val Gln Asn Glu Glu Met Ala Ala Phe 370 375 380 Cys Arg Ser Ile Thr Lys Leu Lys Thr Lys Phe Pro Tyr Thr Asn His 385 390 395 400 Arg Thr Asn Pro Gly Tyr Leu Leu Ser Pro Val Thr Ala Gln Arg Asn 405 410 415 Ile Cys Gly Glu Asn Ala Ser Val Lys Val Ser Ile Asp Ile Glu Gly 420 425 430 Phe Gln Leu Pro Val Thr Phe Thr Cys Asp Val Ser Ser Thr Val Glu 435 440 445 Ile Ile Ile Met Gln Ala Leu Cys Trp Val His Asp Asp Leu Asn Gln 450 455 460 Val Asp Val Gly Ser Tyr Val Leu Lys Val Cys Gly Gln Glu Glu Val 465 470 475 480 Leu Gln Asn Asn His Cys Leu Gly Ser His Glu His Ile Gln Asn Cys 485 490 495 Arg Lys Trp Asp Thr Glu Ile Arg Leu Gln Leu Leu Thr Phe Ser Ala 500 505 510 Met Cys Gln Asn Leu Ala Arg Thr Ala Glu Asp Asp Glu Thr Pro Val 515 520 525 Asp Leu Asn Lys His Leu Tyr Gln Ile Glu Lys Pro Cys Lys Glu Ala 530 535 540 Met Thr Arg His Pro Val Glu Glu Leu Leu Asp Ser Tyr His Asn Gln 545 550 555 560 Val Glu Leu Ala Leu Gln Ile Glu Asn Gln His Arg Ala Val Asp Gln 565 570 575 Val Ile Lys Ala Val Arg Lys Ile Cys Ser Ala Leu Asp Gly Val Glu 580 585 590 Thr Leu Ala Ile Thr Glu Ser Val Lys Lys Leu Lys Arg Ala Val Asn 595 600 605 Leu Pro Arg Ser Lys Thr Ala Asp Val Thr Ser Leu Phe Gly Gly Glu 610 615 620 Asp Thr Ser Arg Ser Ser Thr Arg Gly Ser Leu Asn Pro Glu Asn Pro 625 630 635 640 Val Gln Val Ser Ile Asn Gln Leu Thr Ala Ala Ile Tyr Asp Leu Leu 645 650 655 Arg Leu His Ala Asn Ser Gly Arg Ser Pro Thr Asp Cys Ala Gln Ser 660 665 670 Ser Lys Ser Val Lys Glu Ala Trp Thr Thr Thr Glu Gln Leu Gln Phe 675 680 685 Thr Ile Phe Ala Ala His Gly Ile Ser Ser Asn Trp Val Ser Asn Tyr 690 695 700 Glu Lys Tyr Tyr Leu Ile Cys Ser Leu Ser His Asn Gly Lys Asp Leu 705 710 715 720 Phe Lys Pro Ile Gln Ser Lys Lys Val Gly Thr Tyr Lys Asn Phe Phe 725 730 735 Tyr Leu Ile Lys Trp Asp Glu Leu Ile Ile Phe Pro Ile Gln Ile Ser 740 745 750 Gln Leu Pro Leu Glu Ser Val Leu His Leu Thr Leu Phe Gly Ile Leu 755 760 765 Asn Gln Ser Ser Gly Ser Ser Pro Asp Ser Asn Lys Gln Arg Lys Gly 770 775 780 Pro Glu Ala Leu Gly Lys Val Ser Leu Pro Leu Cys Asp Phe Arg Arg 785 790 795 800 Phe Leu Thr Cys Gly Thr Lys Leu Leu Tyr Leu Trp Thr Ser Ser His 805 810 815 Thr Asn Ser Val Pro Gly Thr Val Thr Lys Lys Gly Tyr Val Met Glu 820 825 830 Arg Ile Val Leu Gln Val Asp Phe Pro Ser Pro Ala Phe Asp Ile Ile 835 840 845 Tyr Thr Thr Pro Gln Val Asp Arg Ser Ile Ile Gln Gln His Asn Leu 850 855 860 Glu Thr Leu Glu Asn Asp Ile Lys Gly Lys Leu Leu Asp Ile Leu His 865 870 875 880 Lys Asp Ser Ser Leu Gly Leu Ser Lys Glu Asp Lys Ala Phe Leu Trp 885 890 895 Glu Lys Arg Tyr Tyr Cys Phe Lys His Pro Asn Cys Leu Pro Lys Ile 900 905 910 Leu Ala Ser Ala Pro Asn Trp Lys Trp Gly Asn Leu Ala Lys Thr Tyr 915 920 925 Ser Leu Leu His Gln Trp Pro Ala Leu Tyr Pro Leu Ile Ala Leu Glu 930 935 940 Leu Leu Asp Ser Lys Phe Ala Asp Gln Glu Val Arg Ser Leu Ala Val 945 950 955 960 Thr Trp Ile Glu Ala Ile Ser Asp Asp Glu Leu Thr Asp Leu Leu Pro 965 970 975 Gln Phe Val Gln Ala Leu Lys Tyr Glu Ile Tyr Leu Asn Ser Ser Leu 980 985 990 Val Gln Phe Leu Leu Ser Arg Ala Leu Gly Asn Ile Gln Ile Ala His 995 1000 1005 Asn Leu Tyr Trp Leu Leu Lys Asp Ala Leu His Asp Val Gln Phe 1010 1015 1020 Ser Thr Arg Tyr Glu His Val Leu Gly Ala Leu Leu Ser Val Gly 1025 1030 1035 Gly Lys Arg Leu Arg Glu Glu Leu Leu Lys Gln Thr Lys Leu Val 1040 1045 1050 Gln Leu Leu Gly Gly Val Ala Glu Lys Val Arg Gln Ala Ser Gly 1055 1060 1065 Ser Ala Arg Gln Val Val Leu Gln Arg Ser Met Glu Arg Val Gln 1070 1075 1080 Ser Phe Phe Gln Lys Asn Lys Cys Arg Leu Pro Leu Lys Pro Ser 1085 1090 1095 Leu Val Ala Lys Glu Leu Asn Ile Lys Ser Cys Ser Phe Phe Ser 1100 1105 1110 Ser Asn Ala Val Pro Leu Lys Val Thr Met Val Asn Ala Asp Pro 1115 1120 1125 Leu Gly Glu Glu Ile Asn Val Met Phe Lys Val Gly Glu Asp Leu 1130 1135 1140 Arg Gln Asp Met Leu Ala Leu Gln Met Ile Lys Ile Met Asp Lys 1145 1150 1155 Ile Trp Leu Lys Glu Gly Leu Asp Leu Arg Met Val Ile Phe Lys 1160 1165 1170 Cys Leu Ser Thr Gly Arg Asp Arg Gly Met Val Glu Leu Val Pro 1175 1180 1185 Ala Ser Asp Thr Leu Arg Lys Ile Gln Val Glu Tyr Gly Val Thr 1190 1195 1200 Gly Ser Phe Lys Asp Lys Pro Leu Ala Glu Trp Leu Arg Lys Tyr 1205 1210 1215 Asn Pro Ser Glu Glu Glu Tyr Glu Lys Ala Ser Glu Asn Phe Ile 1220 1225 1230 Tyr Ser Cys Ala Gly Cys Cys Val Ala Thr Tyr Val Leu Gly Ile 1235 1240 1245 Cys Asp Arg His Asn Asp Asn Ile Met Leu Arg Ser Thr Gly His 1250 1255 1260 Met Phe His Ile Asp Phe Gly Lys Phe Leu Gly His Ala Gln Met 1265 1270 1275 Phe Gly Ser Phe Lys Arg Asp Arg Ala Pro Phe Val Leu Thr Ser 1280 1285 1290 Asp Met Ala Tyr Val Ile Asn Gly Gly Glu Lys Pro Thr Ile Arg 1295 1300 1305 Phe Gln Leu Phe Val Asp Leu Cys Cys Gln Ala Tyr Asn Leu Ile 1310 1315 1320 Arg Lys Gln Thr Asn Leu Phe Leu Asn Leu Leu Ser Leu Met Ile 1325 1330 1335 Pro Ser Gly Leu Pro Glu Leu Thr Ser Ile Gln Asp Leu Lys Tyr 1340 1345 1350 Val Arg Asp Ala Leu Gln Pro Gln Thr Thr Asp Ala Glu Ala Thr 1355 1360 1365 Ile Phe Phe Thr Arg Leu Ile Glu Ser Ser Leu Gly Ser Ile Ala 1370 1375 1380 Thr Lys Phe Asn Phe Phe Ile His Asn Leu Ala Gln Leu Arg Phe 1385 1390 1395 Ser Gly Leu Pro Ser Asn Asp Glu Pro Ile Leu Ser Phe Ser Pro 1400 1405 1410 Lys Thr Tyr Ser Phe Arg Gln Asp Gly Arg Ile Lys Glu Val Ser 1415 1420 1425 Val Phe Thr Tyr His Lys Lys Tyr Asn Pro Asp Lys His Tyr Ile 1430 1435 1440 Tyr Val Val Arg Ile Leu Trp Glu Gly Gln Ile Glu Pro Ser Phe 1445 1450 1455 Val Phe Arg Thr Phe Val Glu Phe Gln Glu Leu His Asn Lys Leu 1460 1465 1470 Ser Ile Ile Phe Pro Leu Trp Lys Leu Pro Gly Phe Pro Asn Arg 1475 1480 1485 Met Val Leu Gly Arg Thr His Ile Lys Asp Val Ala Ala Lys Arg 1490 1495 1500 Lys Ile Glu Leu Asn Ser Tyr Leu Gln Ser Leu Met Asn Ala Ser 1505 1510 1515 Thr Asp Val Ala Glu Cys Asp Leu Val Cys Thr Phe Phe His Pro 1520 1525 1530 Leu Leu Arg Asp Glu Lys Ala Glu Gly Ile Ala Arg Ser Ala Asp 1535 1540 1545 Ala Gly Ser Phe Ser Pro Thr Pro Gly Gln Ile Gly Gly Ala Val 1550 1555 1560 Lys Leu Ser Ile Ser Tyr Arg Asn Gly Thr Leu Phe Ile Met Val 1565 1570 1575 Met His Ile Lys Asp Leu Val Thr Glu Asp Gly Ala Asp Pro Asn 1580 1585 1590 Pro Tyr Val Lys Thr Tyr Leu Leu Pro Asp Asn His Lys Thr Ser 1595 1600 1605 Lys Arg Lys Thr Lys Ile Ser Arg Lys Thr Arg Asn Pro Thr Phe 1610 1615 1620 Asn Glu Met Leu Val Tyr Ser Gly Tyr Ser Lys Glu Thr Leu Arg 1625 1630 1635 Gln Arg Glu Leu Gln Leu Ser Val Leu Ser Ala Glu Ser Leu Arg 1640 1645 1650 Glu Asn Phe Phe Leu Gly Gly Val Thr Leu Pro Leu Lys Asp Phe 1655 1660 1665 Asn Leu Ser Lys Glu Thr Val Lys Trp Tyr Gln Leu Thr Ala Ala 1670 1675 1680 Thr Tyr Leu 1685 3 27 DNA Artificial Sequence primer 3 ctcttcctca tggtctaata cctccac 27 4 20 DNA Artificial Sequence primer 4 ggngangann tncgncanga 20 5 27 DNA Artificial Sequence primer 5 naantgnccn aantcnatnt gntgnaa 27 6 27 DNA Artificial Sequence primer 6 tatctccaaa tcagtccttg ctttccc 27 7 147 PRT Homo sapiens 7 Met Pro Thr Phe Pro Ser Thr Glu Ser Val Tyr Leu Arg Leu Pro Gly 1 5 10 15 Gln Ser Pro Tyr Phe Ser Tyr Pro Leu Thr Pro Ala Thr Pro Phe His 20 25 30 Pro Gln Gly Ser Leu Pro Val Tyr Arg Pro Leu Val Ser Pro Asp Met 35 40 45 Ala Lys Leu Phe Glu Lys Ile Ala Ser Thr Ser Glu Phe Leu Lys Asn 50 55 60 Gly Lys Ala Arg Thr Asp Leu Glu Ile Ala Asn Ser Lys Ala Ser Val 65 70 75 80 Cys Asn Leu Gln Ile Ser Pro Lys Ser Glu Asp Ile Asn Lys Phe Asp 85 90 95 Trp Leu Asp Leu Asp Pro Leu Ser Lys Pro Lys Val Asp Tyr Val Glu 100 105 110 Val Leu Glu His Glu Glu Glu Lys Lys Asp Pro Val Leu Leu Ala Glu 115 120 125 Asp Pro Trp Asp Ala Val Leu Leu Glu Glu Arg Ser Pro Ser Cys His 130 135 140 Leu Glu Arg 145 8 294 PRT Homo sapiens 8 Met Ala Gln Ile Ser Asn Asn Ser Glu Phe Lys Gln Cys Ser Ser Ser 1 5 10 15 His Pro Glu Pro Ile Arg Thr Lys Asp Val Asn Lys Ala Glu Ala Leu 20 25 30 Gln Met Glu Ala Glu Ala Leu Ala Lys Leu Gln Lys Asp Arg Gln Met 35 40 45 Thr Asp Ser Pro Arg Gly Phe Glu Leu Ser Ser Ser Thr Arg Gln Arg 50 55 60 Thr Gln Gly Phe Asn Lys Gln Asp Tyr Asp Leu Met Val Phe Pro Glu 65 70 75 80 Leu Asp Ser Gln Lys Arg Ala Val Asp Ile Asp Val Glu Lys Leu Thr 85 90 95 Gln Ala Glu Leu Glu Lys Ile Leu Leu Asp Asp Asn Phe Glu Thr Arg 100 105 110 Lys Pro Pro Ala Leu Pro Val Thr Pro Val Leu Ser Pro Ser Phe Ser 115 120 125 Thr Gln Leu Tyr Leu Arg Pro Ser Gly Gln Arg Gly Gln Trp Pro Pro 130 135 140 Gly Leu Cys Gly Pro Ser Thr Tyr Thr Leu Pro Ser Thr Tyr Pro Ser 145 150 155 160 Ala Tyr Ser Lys Gln Ala Thr Phe Gln Asn Gly Phe Ser Pro Arg Met 165 170 175 Pro Thr Phe Pro Ser Thr Glu Ser Val Tyr Leu Arg Leu Pro Gly Gln 180 185 190 Ser Pro Tyr Phe Ser Tyr Pro Leu Thr Pro Ala Thr Pro Phe His Pro 195 200 205 Gln Gly Ser Leu Pro Val Tyr Arg Pro Leu Val Ser Pro Asp Met Ala 210 215 220 Lys Leu Phe Glu Lys Ile Ala Ser Thr Ser Glu Phe Leu Lys Asn Gly 225 230 235 240 Lys Ala Arg Thr Asp Leu Glu Ile Ala Asn Ser Lys Ala Ser Val Cys 245 250 255 Asn Leu Gln Ile Ser Pro Lys Ser Glu Asp Ile Asn Lys Phe Asp Trp 260 265 270 Leu Asp Leu Asp Pro Trp Asp Ala Val Leu Leu Glu Glu Arg Ser Pro 275 280 285 Ser Cys His Leu Glu Arg 290 9 7 PRT Homo sapiens MISC_FEATURE (7)..(7) Xaa can be Asp or Glu 9 Gly Asp Asp Leu Arg Gly Xaa 1 5 10 8 PRT Homo sapiens 10 Phe His Ile Asp Phe Gly His Phe 1 5 11 6 PRT Homo sapiens 11 Pro Pro Leu Pro Pro Arg 1 5 

What is claimed is:
 1. A method for identifying a ligand which modulates kinase activity of a class II PI 3 kinase, said method comprising the steps of: i) exposing a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a ligand-binding fragment thereof to at least one ligand; and ii) determining the binding activity of said at least one ligand with respect to said polypeptide.
 2. A method according to claim 1, wherein said polypeptide comprises amino acids 1-337 of SEQ ID NO:
 2. 3. A method according to claim 1, wherein said polypeptide comprises amino acids 1-176 of SEQ ID NO:
 2. 4. A method according to claim 1, wherein said polypeptide is expressed by a cell-line which has been transformed or transfected with a nucleic acid comprising a nucleic acid sequence which hybridizes to SEQ ID NO:1 in 0.5 M sodium phsophate, pH 7.2, 7% SDS, 1 mM EDTA at 65° C. and remains bound after two washing steps with 0.5 x SSC and 0.1% SDS for 20 minutes at 60° C.
 5. A method according to claim 4, wherein said cell line is a prokaryotic cell line.
 6. A method according to claim 4, wherein said cell line is a eukaryotic cell line.
 7. A method according to claim 6, wherein said cell-line is a mammalian cell line.
 8. A method according to claim 7, wherein said mammalian cell line is a kidney cell line.
 9. A method according to claim 4, wherein said nucleic acid is a recombinant vector which comprises: i) a nucleotide sequence which encodes a class II PI3 kinase having the amino acid sequence of SEQ ID NO:2 or a ligand-binding fragment thereof; and ii) a DNA promoter element which is functionally linked to said nucleotide sequence.
 10. A method according to claim 1, wherein said ligand is a peptide or a polypeptide.
 11. A method according to claim 1, wherein said ligand binds the amino-terminal domain of SEQ ID NO:2.
 12. A method according to claim 11, wherein said amino-terminal domain comprises amino acids 1-337 of SEQ ID NO:2.
 13. A method according to claim 11, wherein said amino-terminal domain comprises amino acids 1-176 of SEQ ID NO:2.
 14. A method according to claim 1, wherein said ligand is a class II PI 3 kinase antagonist.
 15. A method according to claim 1, wherein said ligand is a class II PI 3 kinase agonist.
 16. A method according to claim 1, wherein said binding activity modulates the kinase activity of a polypeptide comprising SEQ ID NO:2.
 17. A method according to claim 16, wherein the modulation affects addition of phosphate to a substrate of said enzyme.
 18. A method according to claim 17, wherein said substrate is selected from the group consisting of inositol and PtdIns.
 19. A method according to claim 17, wherein said substrate is PtdIns or PtdIns(4)P.
 20. A method according to claim 16, wherein the modulation of kinase activity by said ligand results in an inhibition of cell division.
 21. A method according to claim 20, wherein the cell is a neutrophil.
 22. A method according to claim 16, wherein the modulation of kinase activity by said ligand results in a stimulation of cell division.
 23. A method according to claim 22, wherein the cell is a neutrophil.
 24. An isolated ligand having class II PI 3 kinase binding activity obtained by: i) exposing a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or a ligand-binding fragment thereof to at least one ligand; ii) determining whether said at least one ligand binds to said polypeptide; and iii) isolating a ligand which binds to said polypeptide.
 25. An isolated ligand according to claim 24, wherein said ligand is a class II PI 3 kinase antagonist.
 26. A ligand according to claim 24, wherein said ligand is a class II PI 3 kinase agonist.
 27. A method according to claim 1, further comprising: preparing a computer aided reconstruction of the 3-dimensional structure of the polypeptide comprising the amino acid sequence of SEQ ID NO:2; and comparing the 3-dimensional structure to the structure of at least one candidate ligand to predict the interaction of the candidate ligand with said polypeptide.
 28. A method according to claim 27, wherein said polypeptide comprises amino acids 1-337 of SEQ ID NO:2.
 29. A method according to claim 27, wherein said polypeptide comprises amino acids 1-176 of SEQ ID NO:2. 